| Light signal and the elongation and dynamics of microtubules play important roles in plant growth and development.SPR1 is a plant specific microtubule associated protein.Previous studies have shown thatSPR1 is involved in regulating cell growth and responsed to abiotic stress.Currently,the research onSPR1 is only deeper in Arabidopsis.However,the function ofSPR1 in woody plant and its regulation on microtubules are not clear,especially whetherSPR1 is involved in light-mediated microtubule elongation and cell expansion remains poorly understood.In this study,the gene of SmSPR1 from Salix matsudana Koidz was used as the research object to investigate the function of SmSPR1 in light-related microtubule elongation and cell expansion.We achieved the following results:(1)SmSPR1 promoter analysis revealed that SmSPR1 was mainly expressed in rapidly growing cells.SmSPR1-GFP location analysis showed that SmSPR1 was mainly located in the nucleus and around cell membrane.Immunofluorescence and co-immunoprecipitation experiments showed that SmSPR1 was colocalized with microtubules.Pharmacological analysis of microtubule depolymerization showed that SmSPR1 had the characteristic of stabilizing microtubules.Overexpression of SmSPR1 resulted in increased tolerance to the microtubule drug of PPM(propyzamide).(2)SPR1 has functional consistency in plants.The gene of SmSPR1 was transferred into poplar and Arabidopsis.The result showed that transgenic poplar and transgenic Arabidopsis were more tolerant to PPM compared with the control group.AtSPR1 was transferred and overexpressed in Arabidopsis,and the phenotype of transgenic seedlings was similar to that of SmSPR1 overexpression seedlings.Moreover,the results of the mutant complementary experiments showed that SmSPR1 could restore the phenotype of spr1 mutant,revealing thatSPR1 has similar biological functions in plants.(3)The influence of SmSPR1 on plant morphology is related to light.The study on the function of SmSPR1 in vivo showed that SmSPR1 had different functions under light and dark conditions.A comparative analysis showed that there was no difference in phenotype between the wild-type and SmSPR1 transgenic plants under light,but in the dark,overexpression of SmSPR1 resulted in right-handed growth of the etiolated hypocotyls.The transgenic etiolated hypocotyls were resumed vertical growth,when turned into the light.It was further found that the microtubule arrangement of SmSPR1 transgenic hypocotyl was similar to that of wild-type under light.However,the anisotropic growth of SmSPR1 transgenic hypocotyl in the dark was weakened,and the microtubule arrangement showed a left-handed spiral,indicating that SmSPR1 participated in the rearrangement of microtubule during the light-dark conversion process.Comparative analysis of gene expression in wild-type and SmSPR1 transgenic plants,it was found that the expression of 234 genes in SmSPR1 transgenic etiolated seedlings were significantly changed in the dark,most of which were involved in light induction,chlorophyll synthesis and photosystem,indicating that these genes may directly or indirectly participate in light-induced plant development.In this study,the proteins of CSN5A(the subunit 5a of CSN)and HY5(ELONGATED HYPOCOTYL 5)were screened and verified by Y2H(Yeast two-hybrid)and Bi FC(bimolecular fluorescence complementation)assays,indicating that these two proteins cooperate with SmSPR1 to participate in the regulation of microtubule arrangement and dynamics.HY5 binds to COP1 in the dark and its function is inhibited.Therefore,SPR1,CSN,and HY5 could interact with each other in the light.In the dark,SPR1 can only be combined with CSN and EB1 to regulate the arrangement and dynamics of microtubule.This study proved and revealed that SmSPR1 participates in light-mediated cell expansion by regulating the arrangement of microtubule,which lays the foundation for further studying the molecular mechanism ofSPR1 involved in plant morphogenesis. |
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